以sbe A和B为靶标的RNAi载体构建及对马铃薯的转化
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摘要
马铃薯(Solanum tuberosum L.)淀粉是一种重要的食品和工业原料。尤其是马铃薯直链淀粉,因其特殊的理化性质而被广泛应用。然而,目前生产上推广的马铃薯栽培品种的直链淀粉含量仅为总淀粉含量的17%,因此,培育高直链淀粉品种对于扩大马铃薯的应用范围和提高其经济价值将具有重要的意义。
马铃薯高直链淀粉材料的培育主要是通过品种间杂交和促进芽变来筛选突变体,但由于马铃薯栽培种为同源四倍体,其染色体杂合性较高和基因分离复杂,选择较为困难,同时受倍性差异的限制,栽培种无法与一些具有高直链淀粉的野生种质(75%左右为二倍体)进行有性杂交,限制了野生种质在作物改良上的应用。
本研究采用RNA干扰技术(RNA interfence,RNAi),以淀粉分支酶(Starch Branching Enzyme,SBE)基因sbe A和sbe B为靶标进行同时干扰,以期待选育出马铃薯高直链淀粉的新品系,在生产上有其积极的生态意义和经济意义。实验结果如下:
1.克隆到淀粉分支酶(SBE)基因: sbe A和sbe B,的部分片段。测序结果表明,克隆的sbe A序列大小为415bp,该序列与GenBank中已公布的序列的同源性为99%。克隆的sbe B序列大小为302bp,该序列与GenBank中已公布的序列的同源性为99%。之后的亚克隆成功得到sbe A和sbe B的融合基因,并引入了适当的酶切位点。
2.克隆到烟草drepp 1基因内含子片段。测序结果表明,克隆的片段大小为300bp,该序列与GeneBank中公布的序列同源性达94%。这段非编码序列为ihpRNA的间隔区,在植物体内转录时形成发卡结构,从而大大提高了沉默效率。
3.构建了以CaMV 35S组成型启动子驱动的含有‘正向sbe融合片段-drepp 1内含子-反向sbe融合片段’的植物表达载体pPSZIF,并将该载体导入根癌农杆菌LBA4404。
4.在遗传转化过程中,先对受体再生体系建立进行了研究。选用‘陇薯3号’、‘Favorita’和‘Shapody’的茎段、试管薯薄片作为外植体材料,以MS为培养基基本成分,对生长激素的浓度和配比进行了优化组合,筛选出有利于受体材料高效再生的培养基配方。最终确定了有利于茎段愈伤诱导的培养基为: MS+2.0 mg/L 6-BA+0.2 mg/L NAA(陇薯3号’、‘Favorita’);MS+2.0 mg/L 6-BA+0.4 mg/L NAA (‘Shapody’)本实验还进行了农杆菌介导马铃薯淀粉分支酶RNAi片段转化马铃薯的研究,初步建立了三个不同品种的马铃薯茎段的再生体系并优化了遗传转化体系。
5.通过外植体Kan敏感性试验,确定了马铃薯茎段、薯片筛选的Kan使用浓度,茎段为50mg/L、薯片为25 mg/L为宜。通过预培养时间、共培养时间对转化的影响研究,建立和优化了转化体系。茎段的预培养2天,共培养时间茎段为2天、薯片以1天为宜。
Potato (Solanum tuberosum L.) starch is one of the most important plant raw materials for both food and industrial applications. Especially, High-amylose starch is in great demand by the starch industry for its unique physicochemical properties. However, so far the amylose content of the potato cultivation species accounts for 17% of total starch content. Therefore cultivate high-amylose potato species will extend the potato application and value.
In the past, the search for high-amylose potato was mainly achieved by extensive breeding programs and the characterization of mutant varieties.Potato has been very difficult to work efficiently using mutational breeding due to the tetraploid nature and hexaploidity.
RNA interference technique (RNAi) is a powerful tool to crop genetic improvement. In order to make sbe A and sbe B gene post-transcriptional gene silencing, we do some work:
The sbe A gene was amplified by RT-PCR from potato tubers’mRNA. The nucleotide sequence of the clone was confirmed by sequencing, the homology between this 415 bp fragment and the corresponding reported sequence is 99%. The sbe B gene was amplified by RT-PCR from potato tubers mRNA. The nucleotide sequence of the clone was confirmed by sequencing, the homology between this 302 bp fragment and the corresponding reported sequence is 99%. Through sub-cloning, we put this two fragment together.
The drepp 1 gene was amplified from tobacco DNA. The nucleotide sequence of the clone was confirmed by sequencing, the homology between this 300 bp fragment and the corresponding reported sequence is 94%. As the partition area, this 300 bp fragment will increase the efficiency of post-transcriptional gene silencing. Then by introducing sbe A and sbe B gene into plasmid pB13, we constructed the sbe-RNAi plant expression vector with CaMV 35S promoter, and the plant expression vector was integrated into agrobaterium LBA4404.
The potato cultivar‘Longshu 3’、‘Favorita’and‘Shapody’were used as plant material to establish the efficient regeneration system by choosing different mediums. At the same time, an efficient transformation system was developed,including the pre-culture of explants, the time of transformation and co-culture. The results indicated that: The optimal duration of pre-culture was 2 days that would improve the rate of stem cutting transformation. The duration of 2 days co-culture might improve the rate of stem cutting transformation. The duration of 1 days co-culture would improve the rate of potato slice transformation.
马铃薯高直链淀粉材料的培育主要是通过品种间杂交和促进芽变来筛选突变体,但由于马铃薯栽培种为同源四倍体,其染色体杂合性较高和基因分离复杂,选择较为困难,同时受倍性差异的限制,栽培种无法与一些具有高直链淀粉的野生种质(75%左右为二倍体)进行有性杂交,限制了野生种质在作物改良上的应用。
本研究采用RNA干扰技术(RNA interfence,RNAi),以淀粉分支酶(Starch Branching Enzyme,SBE)基因sbe A和sbe B为靶标进行同时干扰,以期待选育出马铃薯高直链淀粉的新品系,在生产上有其积极的生态意义和经济意义。实验结果如下:
1.克隆到淀粉分支酶(SBE)基因: sbe A和sbe B,的部分片段。测序结果表明,克隆的sbe A序列大小为415bp,该序列与GenBank中已公布的序列的同源性为99%。克隆的sbe B序列大小为302bp,该序列与GenBank中已公布的序列的同源性为99%。之后的亚克隆成功得到sbe A和sbe B的融合基因,并引入了适当的酶切位点。
2.克隆到烟草drepp 1基因内含子片段。测序结果表明,克隆的片段大小为300bp,该序列与GeneBank中公布的序列同源性达94%。这段非编码序列为ihpRNA的间隔区,在植物体内转录时形成发卡结构,从而大大提高了沉默效率。
3.构建了以CaMV 35S组成型启动子驱动的含有‘正向sbe融合片段-drepp 1内含子-反向sbe融合片段’的植物表达载体pPSZIF,并将该载体导入根癌农杆菌LBA4404。
4.在遗传转化过程中,先对受体再生体系建立进行了研究。选用‘陇薯3号’、‘Favorita’和‘Shapody’的茎段、试管薯薄片作为外植体材料,以MS为培养基基本成分,对生长激素的浓度和配比进行了优化组合,筛选出有利于受体材料高效再生的培养基配方。最终确定了有利于茎段愈伤诱导的培养基为: MS+2.0 mg/L 6-BA+0.2 mg/L NAA(陇薯3号’、‘Favorita’);MS+2.0 mg/L 6-BA+0.4 mg/L NAA (‘Shapody’)本实验还进行了农杆菌介导马铃薯淀粉分支酶RNAi片段转化马铃薯的研究,初步建立了三个不同品种的马铃薯茎段的再生体系并优化了遗传转化体系。
5.通过外植体Kan敏感性试验,确定了马铃薯茎段、薯片筛选的Kan使用浓度,茎段为50mg/L、薯片为25 mg/L为宜。通过预培养时间、共培养时间对转化的影响研究,建立和优化了转化体系。茎段的预培养2天,共培养时间茎段为2天、薯片以1天为宜。
Potato (Solanum tuberosum L.) starch is one of the most important plant raw materials for both food and industrial applications. Especially, High-amylose starch is in great demand by the starch industry for its unique physicochemical properties. However, so far the amylose content of the potato cultivation species accounts for 17% of total starch content. Therefore cultivate high-amylose potato species will extend the potato application and value.
In the past, the search for high-amylose potato was mainly achieved by extensive breeding programs and the characterization of mutant varieties.Potato has been very difficult to work efficiently using mutational breeding due to the tetraploid nature and hexaploidity.
RNA interference technique (RNAi) is a powerful tool to crop genetic improvement. In order to make sbe A and sbe B gene post-transcriptional gene silencing, we do some work:
The sbe A gene was amplified by RT-PCR from potato tubers’mRNA. The nucleotide sequence of the clone was confirmed by sequencing, the homology between this 415 bp fragment and the corresponding reported sequence is 99%. The sbe B gene was amplified by RT-PCR from potato tubers mRNA. The nucleotide sequence of the clone was confirmed by sequencing, the homology between this 302 bp fragment and the corresponding reported sequence is 99%. Through sub-cloning, we put this two fragment together.
The drepp 1 gene was amplified from tobacco DNA. The nucleotide sequence of the clone was confirmed by sequencing, the homology between this 300 bp fragment and the corresponding reported sequence is 94%. As the partition area, this 300 bp fragment will increase the efficiency of post-transcriptional gene silencing. Then by introducing sbe A and sbe B gene into plasmid pB13, we constructed the sbe-RNAi plant expression vector with CaMV 35S promoter, and the plant expression vector was integrated into agrobaterium LBA4404.
The potato cultivar‘Longshu 3’、‘Favorita’and‘Shapody’were used as plant material to establish the efficient regeneration system by choosing different mediums. At the same time, an efficient transformation system was developed,including the pre-culture of explants, the time of transformation and co-culture. The results indicated that: The optimal duration of pre-culture was 2 days that would improve the rate of stem cutting transformation. The duration of 2 days co-culture might improve the rate of stem cutting transformation. The duration of 1 days co-culture would improve the rate of potato slice transformation.
引文
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